Apparatus and method for sorting microfluidic particles

ABSTRACT

A single junction sorter for a microfluidic particle sorter, the single-junction sorter comprising: an input channel, configured to receive a fluid containing particles; an output sort channel and an output waste channel, each connected to the input channel for receiving the fluid therefrom; a bubble generator, operable to selectively displace the fluid around a particle to be sorted and thereby to create a transient flow of the fluid in the input channel; and a vortex element, configured to cause a vortex in the transient flow in order to direct the particle to be sorted into the output sort channel.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/832,095, filed 3 Jun. 2022, now U.S. Pat. No. 11,607,688, issued Mar.21, 2023, which is a continuation of U.S. patent application Ser. No.16/603,106 filed 4 Oct. 2019, now U.S. Pat. No. 11,376,592, issued Jul.5, 2022, which is a national stage application under 35 U.S.C. § 371 ofInternational Application No. PCT/GB2018/051027, filed 19 Apr. 2018,which claims priority to Great Britain Patent Application No. 1706205.0,filed 19 Apr. 2017. The above referenced applications are herebyincorporated by reference into the present application in theirentirety.

BACKGROUND

Instruments for particle sorting have widespread uses in biologicalresearch. A major application of particle sorting technology is to sortbiological cells. Instrumentation for sorting cells based onmeasurements of fluorescent labels within the cells is typically knownas fluorescence activated cell sorting (FACS). Other applications ofparticle sorting include the sorting of solid beads or liquid dropletsof one liquid phase in a carrier fluid. For example, aqueous droplets ina non-aqueous carrier fluid can be used to contain cells. Thus, theparticles to be sorted may, for example, be cells, beads, or dropletscontaining further particles.

A new application of cell sorting technology is the production of celltherapies. Many newer cell therapies require the sorting of largenumbers of cells. For example, many new autologous T-cell therapiesrequire sorting of relatively rare subsets of T-lymphocyte cells fromperipheral blood mononuclear cells (PBMCs). Typically, large numbers ofcells must be sorted in a reasonably short amount of time (e.g. 10⁹cells in around an hour), and the desired cells (which may typicallycompose 1-10% of the total PBMCs) must be recovered with high purity,yield and viability. There is currently no sorter technology capable offulfilling these requirements, in the form of either a researchinstrument or a cell therapy manufacturing instrument.

Other disadvantages of current cell sorting instruments are that theyare not suitable for GMP (good manufacturing practice) production oftherapeutic products, since they are not considered ‘safe by design’ forthe operator or patient. This is because the fluid-wetted components aredifficult to separate from the instrument into an enclosed single-useconsumable, and they produce aerosols which may harm the operator.

The solutions described herein provide a particle sorting technologythat is suitable for sorting a large number of cells, at high viability,yield and purity, in a short amount of time, in an enclosed microfluidicchip that may be integrated into a single-use consumable.

Many microfluidic particle sorting technologies have been invented inthe last couple of decades, although few have reached commercialapplication. A common theme is that of a ‘single-junction sorter’, wherean inlet channel bifurcates into two output channels: a ‘sort’ channeland a ‘waste’ channel. Particles entering the inlet channel are focusedinto the centre of the input channel, typically by a hydrodynamicfocusing region, and the outputs are hydrodynamically biased towards thewaste channel, such that the centre streamline flows into the waste. Anactuator, which is positioned at or upstream of the bifurcation point,selectively exerts a force on a desired particle (or on the fluid aroundthe desired particle) in order to move it away from the centrestreamline and into the sort channel. Microfluidic particle sorters withvarious actuators have been demonstrated, for example actuators based onstanding surface acoustic waves, transient surface acoustic waves,piezo-actuated displacement, micromechanical valves, optical tweezers,electrophoresis, dieletrophoresis and thermal vapour bubbles created bylaser absorption or by electrical heating.

The focusing of the particles into the centre of the inlet channel is animportant part of many particle sorters for two reasons: firstly itallows a greater precision of optical measurement of the particles by afocused laser beam; secondly it allows a smaller deflection of theparticle by the actuator to push the particle from waste stream to sortstream. Alternatives to hydrodynamic focusing are acoustic focusing,described by U.S. Pat. No. 7,340,957 and inertial focusing, described byU.S. Pat. No. 9,347,595.

Microfluidic particle sorters employing thermal vapour bubble actuatorshave been demonstrated. The bubbles are created by electrical heating.The thermal vapour bubble actuator is placed within a side channel. Theeffect of the side channel is to focus and amplify the fluiddisplacement caused by the bubble, so that a particle can be sorted bythe transient displacement directly caused by the bubble itself.However, the side channel is disadvantageous in that it complicates themicrofluidic chip, requiring space on the chip and its own inlet or fillport.

In the case of fragile particles, such as biological cells, there is anupper limit on the sort rate that can be achieved with anysingle-junction sorter. This upper limit is of the order of the maximumshear stress that the particle can withstand without damage. Formammalian cells, this rate is around 20,000 s⁻¹. Therefore, nosingle-junction sorter of this format can achieve the specified sortrate of 10⁹ cells in an hour, or 280,000 per second.

The attempt to parallelize particle sorters within a microfluidic chip(in order to increase the sort rate) has encountered formidabletechnological challenges that stem from the need to parallelize theoptical instrumentation to measure the array of parallel sorters on thechip. For example, for laser-illuminated fluorescence measurement in aparallel sorter, the laser foci have to be split or parallelized, andsimultaneously aligned with the array of microfluidic sorters. Then thecollection optics has to be parallelized, either by scanning across thearray or providing an array detector for each emission wavelengthchannel. To collect light from a sorter at high sensitivity requires ahigh-numerical-aperture objective lens. However, an array of parallelsorters on chip occupies a wider field of view than a single channel.Therefore to achieve an equivalent light collection efficiency requiresa proportionally larger and more expensive objective lens, as well aslarger and more expensive filters and other elements of the opticalsystem. A one-dimensional array of sorters makes poor use of thetwo-dimensional field of view of an objective lens. Furthermore, tominimize the lateral dimension of an array of sorters on a microfluidicchip is challenging, since space is used by the input and outputmanifolds, the hydrodynamic focusing region, and the actuator, all ofwhich must be parallelized. In a parallelized microfluidic sorter, allof the individual sorters must work simultaneously at high fidelity,otherwise the purity and yield of the sorted particle population worsensignificantly.

SUMMARY OF INVENTION

According to an aspect of the invention there is provided asingle-junction sorter for a microfluidic particle sorter, thesingle-junction sorter comprising: an input channel, configured toreceive a fluid containing particles; an output sort channel and anoutput waste channel, each connected to the input channel for receivingthe fluid therefrom; a bubble generator, operable to selectivelydisplace the fluid around a particle to be sorted and thereby to createa transient flow of the fluid in the input channel; and a vortexelement, configured to cause a vortex in the transient flow in order todirect the particle to be sorted into the output sort channel.

The vortex element causes a vortex to be created in the transient flow,which is provided by actuation of the bubble generator. The resultantvortex travels downstream with the particle to be sorted and causes adisplacement (i.e. laterally of the flow axis) of the particle towardand into the output sort channel. This displacement is larger than thedisplacement that would be caused by the actuation of the bubblegenerator in the absence of the vortex element, and the vortex elementtherefore obviates the need for a bubble generator provided in a sidechannel. This advantageously allows for single-junction sortersaccording to the present invention to be efficiently parallelized on achip.

As used herein, the word “particle” encompasses biological cells, solidbeads, and liquid droplets of one liquid phase in a carrier fluid (suchas aqueous droplets in a non-aqueous carrier fluid). Liquid droplets maythemselves contain further particles.

As used herein, the word “fluid” encompasses both aqueous andnon-aqueous fluids, typically in the liquid or gas phase. For thepurposes of the present invention, such a fluid typically containsparticles, although fluids not containing particles may also be used.

The skilled person will understand that the terms “particle” and “fluid”are not limited to the above definitions should also be interpretedaccording to their understood meanings in the art.

Throughout this specification, the terms “output sort channel”, “sortoutput channel”, and “sort outlet” are used interchangeably. Similarly,“output waste channel” should be read as interchangeable with “wasteoutput channel” and “waste outlet”.

The vortex element may comprise a protrusion in the input channel. Thevortex element may comprise a turn in the input channel. The vortexelement may comprise a recess in the input channel. The vortex elementmay be between the bubble generator and the output sort channel. It willbe understood that the vortex element may take any shape, form orgeometry which is suitable to provide a vorticial flow for directing theselected particle to the output sort channel.

The bubble generator may comprise a microheater. In this case, the fluidmay be any liquid that is sufficiently volatile for the microheater togenerate a bubble, such as water, an aqueous solution, or a non-aqueouscarrier medium.

The single-junction sorter may be configured, in the non-operation ofthe bubble generator and thereby absence of the said transient flow, todirect the particles into the output waste channel.

The single-junction sorter may comprise an inertial focuser configuredto centralise the particles in the fluid along a centre of the inputchannel. The inertial focuser may comprise a serpentine channel. Theinput channel may comprise the inertial focuser.

Debris may accumulate during operation of a single-junction sorteraccording to an embodiment of the first aspect of the invention. Inorder to address this issue, a single-junction sorter may comprise avalve configured to close to prevent the fluid passing through theoutput sort channel in order to disrupt the flow of the fluid andthereby direct accumulated debris towards the output waste channel.

As used herein, the word “valve” encompasses conventional flow controldevices, such as a normally-open solenoid valve, as well as flowrestrictors capable of selectively substantially stopping the flow inthe sort output channel of the disclosed embodiments.

The valve could be substituted for any sort of flow restriction device,flow restrictor, closure mechanism/means, flow diverting mechanism/meansor blocking mechanism/means that is capable of selectively substantiallystopping the flow in the support output channel in order to directdebris into the waste channel. Furthermore, it is not necessary for thechannel to be completely blocked, so long as the flow is sufficientlyrestricted to disrupt the flow of the fluid and direct accumulateddebris towards the output waste channel.

According to another aspect of the invention there is provided amicrofluidic particle sorter, comprising an array of single-junctionsorters each as described herein above.

The microfluidic particle sorter may comprise an array of microlenses,each microlens being aligned with a respective one of the array ofsingle-junction sorters.

In the microfluidic particle sorter: the input channels of thesingle-junction sorters may be connected to a common inlet via an inletmanifold; the output waste channels of the single-junction sorters maybe connected to a common waste outlet via a waste manifold; and theoutput sort channels of the single-junction sorters may be connected toa common sort outlet via a sort manifold.

The microfluidic particle sorter may comprise an objective lensarrangement including one or more objective lenses. The objective lensarrangement may be configured to deliver light to and collect light fromevery single-junction sorter of the array of single-junction sorters forthe purpose of characterizing the particles in the fluid. Thus, thelight for control of sorting and particle characterization is deliveredand collected through at least one objective lens, covering the wholearea of the two-dimensional array of single-junction sorters, as theobjective lens arrangement is configured to illuminate the whole area ofthe two-dimensional array.

According to an aspect of the invention there is provided a method ofsorting particles using a single-junction sorter as described hereinabove, the method comprising: providing the input channel with a flow offluid containing particles; and operating the bubble generator in orderto selectively displace the fluid around a particle to be sorted,thereby to create a transient flow of the fluid in the input channelwhich encounters the vortex element, so as to cause a vortex in thetransient flow in order to direct the particle to be sorted into theoutput sort channel.

A further aspect of the invention provides a particle sorter comprising:an input channel configured to receive a fluid; an output sort channeland an output waste channel, each connected to the input channel forreceiving fluid therefrom; and a valve configured to close to preventthe fluid passing through the output sort channel in order to disruptthe flow of the fluid and thereby direct accumulated debris towards theoutput waste channel.

The present invention also provides a method of clearing accumulateddebris in a particle sorter of said further aspect of the invention, themethod comprising: directing a fluid into the input channel; and closingthe valve, thereby disrupting the flow of the fluid to direct theaccumulated debris towards the output waste channel.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments will now be described, by way of example, with reference tothe accompanying figures in which:

FIG. 1 shows a two-dimensional array microfluidic sorter chip;

FIG. 2 shows details of the chip construction, including the layerfabrication structure;

FIG. 3 shows details of the first layer of the chip;

FIG. 4 shows detail of the second layer of the chip;

FIG. 5 shows details of the third layer of the chip, views 5 a and 5 bshowing different perspectives the upper face of the third layer, view 5c showing the lower face of the third layer;

FIG. 6 shows details of the fourth layer of the chip from differentperspectives, views 6 a and 6 b showing two perspectives of the upperface of the fourth layer;

FIG. 7 shows details of the thermal bubble actuators in plan andcross-sectional profiles;

FIG. 8 shows details of the electrical connector;

FIG. 9 shows details of the single-junction sorter;

FIG. 10 shows details of the inertial focuser;

FIG. 11 shows details of a second embodiment of the single-junctionsorter;

FIG. 12 shows details of a third embodiment of the single-junctionsorter;

FIG. 13 shows details of an optical measurement apparatus;

FIG. 14 shows details of a control system;

FIG. 15 shows the operation of the third embodiment of thesingle-junction sorter; and

FIG. 16 shows alternative embodiments of the single junction sorter ofthe present invention that allow for multi-way sorting, with views 16(a)and 16(b) showing designs for 3- and 5-way sorting, respectively.

DETAILED DESCRIPTION

The sorter is embodied by a microfluidic chip pictured in FIG. 1 ,having an inlet port 101, and two outlet ports: a waste outlet 102 and asort outlet 103. Downstream of the inlet port is first the inletmanifold 104, followed by the inertial focusing region 105, then thetwo-dimensional array of single-junction sorters 106, then the waste andsort manifolds (107 and 108, overlaid in the diagram), which connect tothe waste and sort outlets. On the edge of the chip is an electricalconnector 109.

The chip construction is detailed in FIG. 2 , and consists of severallayers of different functions. The first layer 201 is a glass sheet ofthickness 300 μm, which seals the chip on the lower face and providesthe substrate for the deposition of thin film features that make thethermal vapour bubble actuator and electrical contacts (describedbelow). The second layer 202 consists of a micromoulded sheet ofpolydimethylsilicone (PDMS) with a thickness of 60 μm, and contains aset of microchannels described below. The third layer 203 consists of amicromoulded layer of cyclic olefin copolymer (COC) of thickness 300 μm,and contains microchannels on one side with a depth of 60 μm, andthrough-layer vias with a length of 240 μm. The fourth layer 204consists of micromoulded COC of thickness 300 μm, and seals the chip onthe upper face. The layers are bonded together using organosilanesurface functionalisation, plasma treatment, thermal fusion andalignment/bonding equipment known in the art.

FIG. 3 shows the design of the first layer 201. The thermal vapourbubble actuators comprise a two-dimensional four-by-four square array ofthin film metal resistors 302, each henceforth referred to as amicroheater. Each microheater is connected by conduction tracks 303 to acontact pad 304, and on the other side to a common ground pad 305.

FIG. 4 shows the design of the second layer 202. The input manifold 104splits into an array of 16 channels of width 100 μm and pitch 250 μm.Each of these channels is the input channel 400 of a single-junctionsorter. The input channel comprises an inertial focusing section whichconsists of a symmetric serpentine channel 401. The input channel thenconnects to a sorter junction 402. The sorter junctions are placed on atwo-dimensional four-by-four square array, which has a pitch of 1 mm inboth directions, so that each sorter junction aligns with a microheaterin the first layer (302, FIG. 3 ). (The exact alignment is detailedbelow.) At the sorter junction, the input channel splits into a wastechannel of width 71 μm and a sort channel of width 56 μm. The wastechannel 404 continues along the chip, where it is part of an array of 16parallel waste channels that join the waste manifold 107. The sortchannel 403 reaches an end point in the second layer, where it continuesthrough a via into the third layer described below.

FIG. 5 shows the design of the third layer 203, and presents aperspective view (FIG. 5 a ) as well as a drawing of the upper face(FIG. 5 b ) and lower face (FIG. 5 c ). The third layer provides thecontinuation of the sort channels. The sort channel end points in thesecond layer are aligned with a two-dimensional four-by-four squarearray of through-layer via holes 501 each of diameter 100 um. The sortchannels continue from the lower face through these vias to an array of16 parallel microchannels 502 on the upper face, each of width 56 μm.The sort channels then join the sort manifold 108. The third layer alsoprovides a via for the input port 503 and a via for the waste output 504that are aligned respectively with the end points of the input and wastemanifolds in the second layer.

FIG. 6 shows the design of the fourth layer 204, and presents a drawingof the upper face (FIG. 6 a ) and a perspective view (FIG. 6 b ). Thefourth layer seals the microchannels of the third layer. It alsoprovides the input port 101, the waste output port 102, and the sortoutput port 103. These ports consist of through-layer vias, which arealigned with vias 503 and 504 and the end point of the sort manifold 108respectively.

Details of the thermal bubble actuators are presented in FIG. 7 ,showing a drawing of the plan view (FIG. 7 a ) and a schematic of thecross sectional profile (FIG. 7 b ). The microheater 302 comprises aconnector section 701, which overlaps with the conduction tracks 303,and a square section 702 of dimension 50×50 μm, which is the active partof the microheater since it does not overlap with any other conductor.The thermal bubble actuator comprises several layers fabricated by thinfilm deposition techniques on top of the glass substrate 201. These arein order: a passivation layer 801 of thickness 150 nm composed ofsilicon nitride, a resistor 302 of thickness 100 nm composed oftitanium, a second passivation layer 802 of thickness 150 nm composed ofsilicon nitride, and an anti-cavitation layer 803 of thickness 250 nmcomposed of tantalum. The conduction tracks are composed of 20 nm ofnickel-chromium and 100 nm of gold.

The thermal bubble actuators are connected via their conduction tracksto the electrical connector 109: the design of this is shown in FIG. 8 .

The details of the individual single-junction sorter are shown in FIG. 9and FIG. 10 . FIG. 9 shows the sorter junction 402, where the inputchannel 400 divides into the sort channel 403 and the waste channel 404.A recess 901 of width 50 μm in the side of the input channel is alignedwith the active part of the microheater 702, and positioned 300 μmupstream of the junction. FIG. 10 shows the inertial focuser 401, whichconsists of a symmetric serpentine channel. Each inertial focuserconsists of 40 circular arcs of alternating direction, each of radius210 μm (at the channel centre line) and arc angle 90°.

A second embodiment comprising only one single-junction sorter is shownin FIG. 11 . The inlet manifold 104 is connected to the inlet channel400, which contains the serpentine inertial focusing section 401. Theinlet channel then reaches the sort junction 402 and splits into thesort channel 403 and waste channel 404. These continue to join with thesort manifold 108 and waste manifold 107 respectively.

A third alternative embodiment of the single-junction sorter is shown inFIG. 12 . It comprises a main inlet channel 400 (dimensions: 100 μmwidth, 60 μm height), with a microheater 702 (dimensions: 100 μm length,40 μm width) situated in a recess 901 off the left wall (viewed lookingdownwards onto the chip, as in the figure). After the recess, thechannel makes a 90° right turn 1201, continues for 100 μm, then makes a90° left turn 1202. Immediately following the left turn, there is asecond triangular recess on the left wall 1203 (with dimensions: 100 μmlength, 40 μm width), such that an acute angle edge 1204 is formedbetween the left turn and second recess. Following the second recess,the channel contains a pinched region 1205 (90 μm width), beforereaching an opened region 1206 (140 μm width), then splitting into twosymmetric 60 μm width channels. The left channel is the sort channel 403and the right channel is the waste channel 404.

The operation of the particle sorter is as follows.

The input particle suspension, which may be, for example, an aqueoussuspension of lymphocytes of typical diameter 8 μm, at a density of upto around 4×10⁶ cells/mL, is supplied to the input port 101 at a rate ofapproximately 5 mL/min. The input manifold 104 splits the suspensionevenly into the 16 input channels 400.

The inertial focuser 401 causes the particles to align accurately in thecentre of the input channel. It is designed to provide flow conditionsas follows for a centre streamline flow velocity that is preferablybetween 1 m/s and 4 m/s, more preferably 2 m/s. For lymphocytes inaqueous suspension, the Dean number of this flow is approximately 20,the channel Reynolds number is around 120, and the particle Reynoldsnumber is in the range 2-5. We have found experimentally thatrepresentative particles in such an inertial focuser spontaneously focusinto the centre of the channel. Further embodiments may employ any kindof particle focuser as an alternative to the inertial focuser 401.Several kinds of particle focuser are known in the art that are able toaccurately align particles with a streamline in a microfluidic channel,for example sheath flow or hydrodynamic focussing, acoustic focussingand dielectrophoretic focussing.

The particle is measured optically by a laser which is focused at 902just upstream of the microheater 702. The optical measurements typicallyinclude fluorescence, forward scattering and back scattering of light,and the optical reader apparatus for their measurement is describedbelow. The preferred embodiment has a single laser focus persingle-junction sorter. However in alternative embodiments, separatelaser foci may be provided in close proximity upstream of themicroheater, e.g. at 903 and 904. A control system evaluates the opticalmeasurements in real time and decides on whether to sort or reject eachindividual particle before it reaches the microheater.

If the decision is to reject the particle, then it carries on in itsstreamline, which passes into the waste channel 404. However, if thedecision is to sort the particle, then the thermal vapour bubbleactuator is activated, causing the particle to pass into the sortchannel 403. The actuation operates as follows: an electrical pulse ofvoltage 20 V and duration 2 μs is applied between the contact pad 304and ground pad 305, so that an electrical current flows and dissipates acontrolled amount of energy at the microheater. The liquid in thechannel adjacent to the microheater is rapidly heated and goes through aphase transition from liquid to gas, forming a microscopic vapour bubblethat expands and collapses in around 10 μs. Thus the microheateractuates a transient displacement of the liquid around the particle.This displacement increases dues to the fluid's own inertia as thedisplaced fluid moves downstream, so that when the particle reaches thesorter junction 402, its lateral displacement is around 20 μm, which issufficiently large to carry the particle into the sort channel insteadof the waste channel.

The waste manifold 107 and sort manifold 108 collect the outputs of the16 single-junction sorters, and carry them to the waste and sort outputports 102 and 103.

The optical reader apparatus for measurement of fluorescence, forwardscatter and back scatter measurements, is detailed in FIG. 13 . A lasersource of light 1301 is used for fluorescence excitation. The beampasses through a two-dimensional four-by-four microlens array 1302 whichforms a two-dimensional pattern of dots which is then imaged onto thechip by lenses 1303 and 1306. Polarizing beam splitter 1304 transmitsonly one polarization of the light which then reflects from thelong-pass dichroic mirror 1305. Lens 1306 focuses the lightsimultaneously onto the four-by-four array of single-junction sorters ofthe microfluidic chip 1307. (Each focus is made at position 902 on theindividual single-junction sorter, as described above.) Back-scatteredlight that is reflected back from the microfluidic chip is collected bythe lens 1306, after which it is reflected by the mirror 1305; then itenters the beam splitter 1304. The polarization orthogonal to theilluminating beam is then reflected and cleaned by polarizer 1308, thenfocused by lens 1309 and detected as a set of individual spots by atwo-dimensional four-by-four array photodetector 1310.

While fluorescence detection may be collected according to alternativeembodiments in epi- and through modes, epifluorescence detection isprovided in this embodiment. The lens 1306 collects light from bothback-scatter and fluorescence. The long-pass dichroic mirror 1305transmits fluorescence light (which has a longer wavelength than theillumination light). This light then passes through a series offluorescence detection modules 1330. Each module is designed to detectwavelengths within a specified band, and transmit longer wavelengths tothe next module. Each fluorescence detection module 1331 has a long-passdichroic mirror 1311, band-pass optical filter 1312, focusing lens 1313and a two-dimensional four-by-four array of photodetectors 1314. Severaldifferent spectral ranges can be detected simultaneously by stackingmodules with the correct choice of long-pass and band-pass filters, asis known in the art.

Forward-scattered light from the microfluidic chip is collected andcollimated by lens 1315, then reflected by long-pass dichroic mirror1316, after which it is filtered by polarizer 1317 to eliminate thedirectly transmitted light. The forward-scattered light then passesthrough dark field mask 1318 which blocks directly transmitted light andselects a band of angles for forward scatter detection. Theforward-scattered light is then focused by lens 1319 and detected with atwo-dimensional four-by-four array of photodetectors 1320.

In addition to the back scatter, forward scatter and fluorescencemeasurements, imaging of the microfluidic chip is provided, to allow forfocusing and alignment of the illumination source onto the chip. Thetransmission imaging uses a second collimated light source 1321 whichhas a wavelength longer than those measured by the fluorescencedetection modules. This light propagates through all the dichroicmirrors 1311, lens 1306, the microfluidic chip 1307, lens 1315, anddichroic mirror 1316. There is then an additional band-pass filter 1322to remove stray light, then lens 1323 focuses the light onto the camera1324. The light source 1321 provides constant illumination or shortpulses triggered from particle detection events to allow monitoring andcontrol over the sorting procedure.

In a further embodiment, the microfluidic chip is integrated with atwo-dimensional microlens array attached to the glass substrate sideopposite to the microchannels. Each lens is aligned with the laser focuspoint 902 on each a single-junction junction sorter. The microlensesserve to increase the efficiency of fluorescence collection from eachsingle-junction sorter.

The sorter's control system is detailed in FIG. 14 and described asfollows. The control system has a signal processing block 1401 persingle-junction sorter, thus making sixteen processing blocks for thefour-by-four parallel microfluidic particle sorter. Each signalprocessing block has six analogue inputs for the four fluorescencechannels, forward scatter and back scatter signals. A multi-channelanalogue-to-digital converter (ADC) digitizes the signals and transmitsthem across a digital interface to a field programmable gate array(FPGA). The FPGA performs a peak detection and characterizationalgorithm, and makes a decision whether to activate the thermal bubbleactuator for that channel to divert a cell. Peak detection andcharacterization algorithms are known in the field of cytometry and notfurther described here. The actuation signal is passed to a block ofparallel drive transistors 1402.

In each signal processing block, an external memory is interfaced with asoft processor in the FPGA, and allows data from the peakcharacterization to be stored until they are required, such as at theend of a run to collect the cells' peak data for analysis. When thesedata are required, the control processor requests and uploads the datafrom each block sequentially. Additionally the control processor is usedto send data to the FPGA such as thresholds and parameters for thepeak-detection algorithm, parameters of the sorting pulse and commandsto control the sorting process.

The effect of the thermal bubble actuation is amplified by the geometryof the single-junction sorter, which is shown by fluid flow simulationsdepicted in FIG. 15 . Here we use the geometry of the third embodimentof the single-junction sorter above to demonstrate the fluid flow (referto FIG. 12 for the geometric features). Due to the substantial inertiaof the fluid (channel Reynolds number of around 120), the right turn1201 and left turn 1202 cause a greater flow into the waste channel 404than the sort channel 403, so that in the absence of a thermal bubbleactuation, particles that approach the junction on the centre streamlinewill leave through the waste channel. However, when the thermal vapourbubble is actuated, the growth and collapse of the bubble rapidlydisplaces the fluid, first away from the microheater and then towardsthe microheater. As the bubble grows, the transient flow causes a vortexto form near the wall of the second recess 1203. At this point in time,the vortex is away from the main flow and has little interaction withthe particles. However, when the bubble collapses, the transient flowcauses a ‘sorting vortex’ 1501 to form around the acute angle edge 1204,which subsequently moves downsteam with the main flow. Because thesorting vortex moves downstream with the particle to be sorted, itcauses a much larger lateral displacement of a particle than the directdisplacement of the particle caused by the thermal vapour bubble alone.Trajectories of a sorted particle 1502 and an unsorted particle 1503 areshown.

Many alternative embodiments also create such a sorting vortex, forexample where a recess, bend or edge is placed in the single-junctionsorter upstream of the sorting junction.

Further alternative embodiments of the single-junction sorter are shownin FIGS. 16(a) and 16(b), to allow for multi-way sorting. Theembodiments shown in FIGS. 16(a) and 16(b) provide for 3- and 5-waysorting, respectively Each of these embodiments comprises a main inletchannel 400, a microheater 702 situated in a first recess 901 off theleft wall (viewed looking downwards onto the chip, as in the figure).After the first recess 901, there is a second recess 1203 on the leftwall, such that an acute angle edge 1204 is formed between the tworecesses 901 and 1203. Following the second recess 1203, the channelcontains a straight region 1601, before splitting into several symmetricchannels 1602 and 1603. The central channel 1603 is the waste channel,and either side are separate sort channels 1602. Any number of sortoutputs could be provided in alternative embodiments. In thesealternative embodiments, the waste channel 1603 will typically beprovided such that the fluid is substantially directed towards thiswaste channel 1603 during equilibrium flow of the fluid.

In operation, a single thermal bubble actuation is employed to displacea particle into any one of the multi-way sort outputs by the followingmethod. The sorting vortex 1501 is characterised by a flow profile thatvaries in position with respect to the flow path: a particle ahead ofthe centre of the vortex is displaced towards the left, while a particlebehind the centre of the vortex is displaced towards the right. Thetotal displacement of a particle depends on the distance from thevortex. Thus, by careful timing of the actuation with respect to theparticle position, the total displacement is calibrated to match thepositions of the respective output channels. The control system is thenprogrammed to give the actuation pulse at a set of time delays thatcorrespond to each of the multi-way sort outputs.

In operation, for many types of particle suspensions, there is atendency for debris to accumulate at the sort junction and clog or blockthe sorter. According to a further embodiment, a valve, such as anormally-open solenoid valve, is placed at (or downstream of) the sortoutlet. This valve is capable of stopping the flow in the sort outputchannel. The technique to unclog the junction is to temporarily closethis valve, which causes the flow to change around the sort junction,thus sweeping any debris into the waste channel. Typically the valve isclosed for between 0.1 and 20 seconds, more typically for around 1second, to have the unclogging effect. The valve can be actuatedperiodically or whenever debris is detected on the junction by using thecamera.

In the case of the multi-way sorting embodiments (FIGS. 16(a) and16(b)), the unclogging mechanism comprises a separate valve on each ofthe sort outputs. In operation, one or more sort outputs are temporarilyclosed to have the unclogging effect.

A valve could also be provided on the waste outlet, in addition to or asan alternative to the valve provided on the sort outlet, such thatdebris is directed towards one or more of the sort outputs when thisvalve is closed.

The valve could be substituted for any sort of flow restriction device,flow restrictor, closure mechanism/means, flow diverting mechanism/meansor blocking mechanism/means that is capable of selectively substantiallystopping the flow in the support output channel in order to directdebris into the waste channel. Furthermore, it is not necessary for thechannel to be completely blocked, so long as the flow is sufficientlyrestricted to disrupt the flow of the fluid and direct accumulateddebris towards the output waste channel.

The present invention provides a microfluidic particle sorter that iscapable of sorting fragile particles (such as biological cells, beads,or droplets containing further particles) at a much higher sort ratethan was hitherto possible. The invention achieves a high sort rate byproviding a single-junction sorter that is suitable to be parallelizedon a microfluidic chip. The single-junction sorters may be arranged onthe chip in a two-dimensional array, which allows an efficient use ofthe field of view of the objective lens. This two-dimensional array isenabled by the design of the single-junction sorter, which allows adense packing on the chip. Each single-junction sorter provides a bubblegenerator (e.g. a thermal vapour bubble generator) without a sidechannel, and a bifurcation of the stream into sort and waste channels.The geometry of the single-junction sorter is chosen so that theactuation of the thermal vapour bubble creates a ‘sorting vortex’, whichtravels downstream with the particle to be sorted, and thus causes amuch larger lateral displacement of a particle than the directdisplacement of the particle caused by a thermal vapour bubble alone.

A plurality of single-junction sorters may be arranged so that theirinput channels branch off a common input manifold, their sort outputchannels combine into a common sort manifold, and their waste outputchannels combine into a common waste manifold. The channel widths of thesingle-junction sorters may be chosen so that each single-junctionsorter experiences the same input flow velocity and the same ratio offluid flowing down the sort and waste outputs when a pressure differenceis exerted between the input and output ports.

It will be understood that the invention has been described in relationto its preferred embodiments and may be modified in many different wayswithout departing from the scope of the invention as defined by theaccompanying claims.

What is claimed is:
 1. A method for sorting particles in a particlesorter, the method comprising: receiving, at an input channel, an inputparticle suspension comprising a fluid and a plurality of particles;aligning the plurality of particles in a streamline of the fluid;evaluating the plurality of particles at a predetermined location andassigning a sort designation or a reject designation for each particleof the plurality of particles; responsive to a sort designation, causinga creation of a transient flow in the fluid, wherein the transient flowis created at a position downstream of the predetermined location;responsive to the transient flow, causing formation of a sorting vortexin the fluid; and directing particles assigned with the sort designationto an output sort channel via the sorting vortex.